Aspergillus diversity in the environments of nosocomial infection cases at a university hospital

Aspergillus species (sp.) that causes opportunistic infections have been increasingly found in human mainly immunosuppressive patients around the world every year. The main objective was to use a rapid and cheap molecular method for monitoring Aspergillus infections and epidemiological approaches. In order to identity Aspergilli species (spp.), a number of molecular methods including restriction fragment length polymorphism (RFLP) have been employed in accordance with ribosomal RNA amplification. The focus of this study — a group of hospitalized patients with clinical and subclinical signs of infection. All of the collected clinical specimens were transported to the medical mycology lab and examined for Aspergillus identification. The environmental specimens were collected from air and surfaces inspected for the Aspergillus within the hospital sources. At first, growth characteristics and microscopic features on mycological media for the identification of Aspergillus sp. were performed. For the confirmation of Aspergillus isolates which similarly found in clinical and environmental sources, molecular method polymerase chain reaction/restriction fragment length polymorphism was carried out. From the mentioned specimens, 102 fungal isolates included Candida spp., Aspergillus spp. and other fungi. Aspergillus flavus (47%), Aspergillus fumigatus (29.4%) and Aspergillus niger (23.5%) all were found as the most common clinical isolates. In addition, Aspergillus isolates from environmental were Aspergillus niger (43.7%), Aspergillus flavus (41.7%), Aspergillus fumigatus (14.6%). Therefore, polymerase chain reaction-restriction fragment length polymorphism with a single restriction enzyme can be very useful in the identification of Aspergillus spp., because of its facility in use, speed, robust, and high sensitivity of diagnosis.


Introduction
Aspergillus sp. falling under opportunistic fungi are often distributed and observed in soil, water, and decomposing vegetation. These species can also be isolated from unfiltered air, ventilation devices, dropped ceiling dirt, and polluted dust released from hospital renovation and construction operations. Aspergillus sp. may also colonize in catheters and implants [1]. As research findings suggested that these fungi underlay hospital-acquired infections (HAI) [2][3][4][5].
Aspergillus sp. are very profuse and most often found in soil, water, air, seed and, food. Some of these species can contribute to the development of a number of diseases including allergic bronco pulmonary disease, invasive infection, nasal sinusitis, mycotic keratitis, and otomycosis [11]. Aspergillus infection are developed through a path formed due to the inhalation of the fungal spores. And also patients with immunodeficient and immunosuppressed systems, pneumonia can be induced by local lung tissue invasion. In addition, the fungus may also spread into the bloodstream and affect various inner organs. Patients suffering from acute, prolonged granulocytopenia, especially those who undergo a bone-marrow transplant are most likely to be at the risk of developing filamentous fungi diseases, especially Aspergillus sp. Besides, patients diagnosed with aspergillosis are most likely to be infected by A. fumigatus and A. flavus which are regarded as the most commonly isolated species [3].
At least 30 species including A. flavus, A. terreus, A. niger, A. nidulans, A.ustus and A.versicolor underlie human diseases, while A. fumigatus is known as the most common cause of invasive aspergillosis. Therefore, choosing a reliable technique for early diagnosis of Aspergillus sp. leading to HAIs is essential and can contribute to monitor diseases --the effective use of epidemiological approaches. The early diagnosis of Aspergillus infections is a challenging task. During the past decades, with the increase in the number of antifungal agents, more drug resistant Aspergillus sp. have been developed [6][7][8]. The use of conventional microbiological, serologic, and imaging techniques do not lead to early diagnosis and effective treatment of Aspergillus infections. Clinical symptoms and signs and CT scan images can be employed to detect invasive diseases. Tissue invasion can be diagnosed by examining biopsy materials which enhances the chance of detecting invasive disease where the blood cultures are normally negative [9]. Besides, a set of clinical and pathological tests normally used for identification are timeconsuming or less effective for routine surveys.
The chance of detecting HAI-inducing Aspergillus sp. can also be increased by employing a set of immunological and molecular tests [3,9]. Molecular techniques which include the restriction fragment length polymorphism (RFLP) developed based on the amplification of ribosomal RNA have been used to recognize Aspergilli spp. and make effective treatment decisions [10]. PCR-RFLP method was employed in this study with a single restriction enzyme to identify Aspergillus sp. isolated from HAI patients and the hospital interior environments. The advantages of this technique are its ease of use, cost-effectiveness, high speed, and its diversity power [6,11,12]. PCR-RFLP which is based on single restriction enzyme MwoI has been proposed previously due to its reliability and accuracy than conventional techniques [13]. The data in this literature is insufficient for different countries with various circumstances. Therefore, this study addresses the identification of Aspergillus sp. in patients referring to a university hospital in Iran.

Clinical and environmental specimens
Most of the clinical specimens collected in this study are from patients suffering from acute infection symptoms hospitalized at a large university hospital. The invasive, acute disease was diagnosed using the clinical symptoms and CT scan images. Colonization of the patients was done by isolating the organism in the culture of specimens such as bronchiolar lavage, sinus discharge, sputum, synovial fluid, and urine. Our target group in this study included all patients with clinical symptoms of fungal infections 48-72 h after hospitalizing in the ward. All specimens were taken to the Medical Mycology Center, UMS University. Standard morphologic techniques were used to diagnose and identify Aspergillus elements as well as the growth characteristics and microscopic features of sabouraud glucose agar 4%. Besides, PCR-RFLP is a molecular method which is employed to identify isolated Aspergillus [13]. Additional specimens were collected for each positive case from places such as hospital interior spaces and equipment including air, surfaces of the walls and coverings, beds, sheets, trolleys, air conditioning, medical equipment and devices, finger touch samples of cases, staffs, and visitors. Air samples were taken by using sabouraud glucose agar 4% (SGA) plates left uncapped and exposed to air flow. Furthermore, the other samples were collected with sterile swabs and then were inoculated on a transport medium such as SGA 4% [12]. All samples including transport cultures were transferred to the Medical Mycology Center, Urmia Medical Sciences University (UMSU).

DNA extraction
Aspergillus mycelia mass was prepared by filtering fungal liquid cultures for 12-24 h. Genomic DNA was taken from isolates by using glass beads in a lysis buffer (1 mM EDTA, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl, 2% Triton X-100, pH=8.0). Then it was processed by the conventional phenol-chloroform method and was also checked by using agarose gel electrophoresis [14].

PCR amplification
Universal primers were used in this study to amplify Aspergillus ITS regions forward Primer: 5'-TCC GTA GGT GAA CCT GCG G -3' and Reverse Primer: 5'-TCC TCC GCT TAT TGAT TAT GC-3'. In addition, PCR assay was performed using 5 µl of the DNA template in a PCR buffer with the reaction volume of 50 µl (20 mM Tris-HCl, pH=8.0), 50 mM KCl, 0.1 mM each of forward and reverse primers for the ITS regions of ribosomal DNA, and 1.5 U of Taq DNA polymerase. All reactions occurred in a thermal cycler (XL model, Bioer, China). PCR program began by denaturing DNA at 95 °C for 5 min followed by 30 cycles. Each cycle includes three steps: denaturation, annealing, and extension steps at 95, 55, and 62 °C lasting 30 s, 1 min, and 5 min, respectively and a final extension at 72 °C for 5 min after the last cycle. Besides, a negative control, i.e., double deionized water (DDW, Merck, Germany), and positive control, DNA template extracted from standard Candida strain: C. albicans (ATCC 10261) (Boiling Phenol-Chloroform method), were employed. Electrophoresis was used to length separate DNA fragments via 1.5% agarose gels in Tris-Borate EDTA buffer and 0.50 mg ethidium bromide per ml. The results were documented using a tarns illuminator gel doc System (Figure 1).

Digestion of PCR products by RFLP method
The restriction fragment length polymorphism technique was employed to produce differential patterns in order to identify Aspergillus spp. [14]. When amplified ITS fragments are digested with the proposed restriction enzyme, MwoI Besides, A. flavus (47%), A. fumigatus (29.4%) and A. niger (23.5%) were found to represent the most frequent clinically isolated Aspergillus sp., in descending order, as shown in Table 1. The black filamentous mold 'Alternaria alternata' was the only non-Candida, non-Aspergillus isolated fungus derived from a synovial sample of a patient suffering from septic arthritis. As it is shown in Figure 2, all identified samples were confirmed using PCR-RFLP technique.
A total number of 256 specimens were collected from finger touches and body surface samples of the patients, hospital employees, and visitors, hospital devices and equipment including beds, floors, walls, trolleys, sinks, medical devices and air samples ( at 37 °C enables differentiating some medically relevant Aspergillus sp. The same process was repeated for all clinical and environmental Aspergillus isolates . For the restricted digestion, 13 µl of each PCR product was directly digested by 5 U (0.5 µl) of the restriction enzyme, 1.5 µl of the 5X buffer, and was incubated at 37 °C for 180 min [15]. The digested PCR products were then treated with electrophoresis in agarose gel (2%) and visualized with gel doc system. In the last stage, Aspergillus sp. were identified in molecular terms by contrasting the electrophoresis bands with differential patterns (Figure 2).

Results
The    The process used to identify the isolated hospital fungi is of high importance as it contributes to select the most effective antifungal therapy. Furthermore, the use of unreliable techniques leading to misdiagnosis and overtreatment may speed up the spread of hospital infections.
Collecting hospital fungi isolates from cases and employees' finger touch samples is important ( Table 2). Aspergillus spp. representing the second most important group of fungi underlying nosocomial infections was sampled from the air and indoor/outdoor places at the hospital. The most contaminated hospital indoor samples were collected from carpets, walls, and also air specimens in the hospital rooms [2]. Aspergillus spp. is inhaled mainly from the spores existing in aerosols suspended in the air. However, the present study did not address the isolation of Aspergillus spore from air samples. Respirable fungal spores can be released from building or construction projects and spread into the environment [1]. Because of their small size, Aspergillus spores can suspend in the air for a long time. Therefore, the use of antiseptic agents (fungicides) on a daily basis as a routine laboratory procedure is recommended.
Our results of Aspergillus isolation are in agreement with the above studies implying that A. fumigatus and A. flavus are the most commonly isolated Aspergillus spp. in the patients with diagnosed aspergillosis. Molecular assays that allow genetic characterization of Aspergillus phenotype diversity are likely to become incorporated into major mycologist guidelines that are widely applicable in clinical practice. In addition, early identification is important for discovering new antifungal drugs to control hospital and environmental infections [21].

Discussion
There are many evidences to support the role of opportunistic fungi that act as main agents underlying in HAIs. For instance, in the time period from 1980 to 1990, Aspergillus spp. was identified as the main factor leading to life-threatening infections in immunocompromised patients [16]. Aspergillus elements are tipically isolated from soil, water, and compost. The spores have also been found in unfiltered air, air conditioning systems, and dust released through hospital renovation and construction activities.
A couple of molecular techniques were employed recently to discover opportunistic fungi such as Candida and Aspergillus sp. [17,18]. As a case in point, the PCR-RFLP technique was employed by Moody and Tyler [10] to analyze interspecies variations of Aspergillus group flavi such as A. flavus, A. parasiticus, and A. nomius. Dendis et al. [19] also used the same technique to detect some pathogenic fungi in febrile neutropenic patients. RFLP was also used with a single restriction enzyme by Mirhendi and team [20] to identify and differentiate pathogenic fungi including Candida spp. The PCR-RFLP method with a single restriction enzyme, MwoI, was also employed to discover Aspergillus spp. The whole molecular identification process including the isolation of the pathogen fungi from a short incubated culture, using DNA extraction, PCR, and RFLP profiles for 6-10 samples takes about several hours (12-18 h). However, the process lasts at least for 1-5 days with traditional methods. Therefore, this technique is faster than classic or morphologic identification approaches.
PCR-RFLP employing MwoI was used in this study as a rapid and reliable technique to identify Aspergillus spp. released from clinical specimens. Accordingly, it was found that over 50% of all samples in the environmental isolates were positive in terms of the presence of at least one opportunistic fungus. The floor coverings, walls, and beds were the most frequent samples that were contaminated by the above fungi. This finding can be attributed to the fact that the patient cases had contacts with the above surfaces frequently and for a long time.